Mitotic stability of a coding DNA sequence-free version of Leishmania major chromosome 1 generated by targeted chromosome fragmentation

The deletion of a 260-kb segment containing all the coding DNA sequences (CDS) of chromosome 1 of Leishmania major Friedlin strain was performed through homologous recombination during a transfection experiment. This allowed the selection of a mutant clone containing a linear extra chromosome sizing 155 kb (XC155). The structure of XC155 was determined by restriction analysis and DNA cloning and sequencing of the gel-purified chromosome: it is made of a 'mirror' inverted duplication of the 'right' end of chromosome 1a (approximately 25 kb at each end), and in its central part of a complex tandem amplification of the linearized transfection vector containing the hygromycin resistance gene (over approximately 105 kb). No sequence of the coding region of chromosome 1 (including the 1.6-kb 'switch' region) was found. By contrast, XC155 contains two large (approximately 13 kb) clusters of tandemly repeated subtelomeric sequences (272-bp 'satellite' DNA) as well as telomeric hexamer repeats. This extra chromosome was found to be mitotically stable after150 generations without selective pressure in vitro. Two sequence elements are considered which may have an effect on mitotic stability and participate to centromeric function in this extra chromosome: the amplification of the input vector and the 272-bp 'satellite' DNA bound by telomeric repeats.